PROJECT SUMMARY/ABSTRACT Investigations from our laboratory have revealed that Notch1 plays a critical role in osteoblastic cell differentiation and function, and its expression in immature osteoblasts causes osteopenia secondary to an inhibitory effect on osteoblastogenesis. Although Notch2 appears to have an important and distinct function from Notch1, most of the work conducted in skeletal cells has examined the effects of Notch1, and there is limited information on the function of Notch2 in the skeleton. Hajdu-Cheney Syndrome (HCS), a devastating disease characterized by acro-osteolysis, osteoporosis and fractures, was recently attributed to gain of function mutations of NOTCH2 leading to NOTCH2 protein stabilization. The aim of the proposed research is to create and study mouse models of HCS, to understand the function of Notch2 in the skeleton and to define the mechanisms involved in HCS. Our specific aims are: 1) To determine the function of Notch2 and characterize the HCS by creating global and conditional mouse models of Notch2HCS mutant activation in skeletal cells. The skeletal phenotype of global and cell lineage-specific Notch2HCS conditional by inversion (COIN) mutants will be compared to that of wild type mice and determined by contact radiography, densitometry, micro CT scanning and histomorphometry. The biomechanical properties of the skeleton from HCS mutant mice will be analyzed; 2) To determine the mechanism responsible for HCS in the skeleton. To this end, mechanisms responsible for the skeletal phenotype will be established, and we will determine whether the Notch canonical signaling pathway is responsible for the effects of Notch2 and the HCS. In addition, we will determine whether Notch2 mRNA and protein are stabilized in cells from Notch2HCS mutants and explain the phenotype observed. Levels of transcriptional and post-transcriptional regulation of genes modified by Notch2HCS mutants will be examined in vitro; and 3) To determine the role of Notch2 target gene(s) in the skeleton and in the HCS. To this end, we will determine whether the Notch2HCS mutant phenotype is secondary to the activation of Hairy-Enhancer of Split (Hes) 1 and exclude the involvement of Hes1 related with YRPW (Hey) 1, 2 and L. Notch2HCS mutants will be studied in the context (or not) of the conditional Hes1 inactivation, and if appropriate Hey inactivation, for changes in their skeletal phenotype determined by CT scanning and histomorphometry and for changes in osteoblastic gene expression.